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Abstract Background and aims Smoking is recognised as an independent risk factor in the development of chronic pancreatitis (CP). Cystic fibrosis transmembrane conductance regulator (CFTR) function and ductal fluid and bicarbonate secretion are also known to be impaired in CP, so it is crucial to understand the relationships between smoking, pancreatic ductal function and the development of CP. Methods We measured sweat chloride (Cl–) concentrations in patients with and without CP, both smokers and non‐smokers, to assess CFTR activity. Serum heavy metal levels and tissue cadmium concentrations were determined by mass spectrometry in smoking and non‐smoking patients. Guinea pigs were exposed to cigarette smoke, and cigarette smoke extract (CSE) was prepared to characterise its effects on pancreatic HCO3– and fluid secretion and CFTR function. We administered cerulein to both the smoking and non‐smoking groups of mice to induce pancreatitis. Results Sweat samples from smokers, both with and without CP, exhibited elevated Cl– concentrations compared to those from non‐smokers, indicating a decrease in CFTR activity due to smoking. Pancreatic tissues from smokers, regardless of CP status, displayed lower CFTR expression than those from non‐smokers. Serum levels of cadmium and mercury, as well as pancreatic tissue cadmium, were increased in smokers. Smoking, CSE, cadmium, mercury and nicotine all hindered fluid and HCO3– secretion and CFTR activity in pancreatic ductal cells. These effects were mediated by sustained increases in intracellular calcium ([Ca2+]i), depletion of intracellular ATP (ATPi) and mitochondrial membrane depolarisation. Conclusion Smoking impairs pancreatic ductal function and contributes to the development of CP. Heavy metals, notably cadmium, play a significant role in the harmful effects of smoking. Key points Smoking and cigarette smoke extract diminish pancreatic ductal fluid and HCO3– secretion as well as the expression and function of CFTR Cd and Hg concentrations are significantly higher in the serum samples of smokers Cd accumulates in the pancreatic tissue of smokers

Alterations to intestinal microbiota are assumed to occur in the pathogenesis of inflammatory bowel disease (IBD). This study aims to analyze the association of fecal microbiota composition, body composition, and lipid characteristics in patients with Crohn’s disease (CD). In our cross-sectional study, patients with CD were enrolled and blood and fecal samples were collected. Clinical and endoscopic disease activity and body composition were assessed and laboratory tests were made. Fecal bacterial composition was analyzed using the shotgun method. Microbiota alterations based on obesity, lipid parameters, and disease characteristics were analyzed. In this study, 27 patients with CD were analyzed, of which 37.0% were obese based on visceral fat area (VFA). Beta diversities were higher in non-obese patients (p < 0.001), but relative abundances did not differ. C. innocuum had a higher abundance at a high cholesterol level than Bacillota (p = 0.001, p = 0.0034). Adlercreutzia, B. longum, and Blautia alterations were correlated with triglyceride levels. Higher Clostridia (p = 0.009) and B. schinkii (p = 0.032) and lower Lactobacillus (p = 0.035) were connected to high VFA. Disease activity was coupled with dysbiotic elements. Microbiota alterations in obesity highlight the importance of gut microbiota in diseases with a similar inflammatory background and project therapeutic options.

Abstract Background In severe acute pancreatitis (AP) the CNS is affected manifesting in neurological symptoms. Earlier research from our laboratory showed blood–brain barrier (BBB) permeability elevation in a taurocholate-induced AP model. Here we aimed to further explore BBB changes in AP using a different, non-invasive in vivo model induced by l-ornithine. Our goal was also to identify whether l-ornithine, a cationic amino acid, has a direct effect on brain endothelial cells in vitro contributing to the observed BBB changes. Methods AP was induced in rats by the intraperitoneal administration of l-ornithine-HCl. Vessel permeability and the gene expression of the primary transporter of l-ornithine, cationic amino acid transporter-1 (Cat-1) in the brain cortex, pancreas, liver and lung were determined. Ultrastructural changes were followed by transmission electron microscopy. The direct effect of l-ornithine was tested on primary rat brain endothelial cells and a triple co-culture model of the BBB. Viability and barrier integrity, including permeability and TEER, nitrogen monoxide (NO) and reactive oxygen species (ROS) production and NF-κB translocation were measured. Fluorescent staining for claudin-5, occludin, ZO-1, β-catenin, cell adhesion molecules Icam-1 and Vcam-1 and mitochondria was performed. Cell surface charge was measured by laser Doppler velocimetry. Results In the l-ornithine-induced AP model vessel permeability for fluorescein and Cat-1 expression levels were elevated in the brain cortex and pancreas. On the ultrastructural level surface glycocalyx and mitochondrial damage, tight junction and basal membrane alterations, and glial edema were observed. l-ornithine decreased cell impedance and elevated the BBB model permeability in vitro. Discontinuity in the surface glycocalyx labeling and immunostaining of junctional proteins, cytoplasmic redistribution of ZO-1 and β-catenin, and elevation of Vcam-1 expression were measured. ROS production was increased and mitochondrial network was damaged without NF-κB, NO production or mitochondrial membrane potential alterations. Similar ultrastructural changes were seen in l-ornithine treated brain endothelial cells as in vivo. The basal negative zeta potential of brain endothelial cells became more positive after l-ornithine treatment. Conclusion We demonstrated BBB damage in the l-ornithine-induced rat AP model suggesting a general, AP model independent effect. l-ornithine induced oxidative stress, decreased barrier integrity and altered BBB morphology in a culture BBB model. These data suggest a direct effect of the cationic l-ornithine on brain endothelium. Endothelial surface glycocalyx injury was revealed both in vivo and in vitro, as an additional novel component of the BBB-related pathological changes in AP.

The pathophysiology of acute pancreatitis (AP) is not well understood, and the disease does not have specific therapy. Tryptophan metabolite L-kynurenic acid (KYNA) and its synthetic analogue SZR-72 are antagonists of the N-methyl-D-aspartate receptor (NMDAR) and have immune modulatory roles in several inflammatory diseases. Our aims were to investigate the effects of KYNA and SZR-72 on experimental AP and to reveal their possible mode of action. AP was induced by intraperitoneal (i.p.) injection of L-ornithine-HCl (LO) in SPRD rats. Animals were pretreated with 75-300 mg/kg KYNA or SZR-72. Control animals were injected with physiological saline instead of LO, KYNA and/or SZR-72. Laboratory and histological parameters, as well as pancreatic and systemic circulation were measured to evaluate AP severity. Pancreatic heat shock protein-72 and IL-1β were measured by western blot and ELISA, respectively. Pancreatic expression of NMDAR1 was investigated by RT-PCR and immunohistochemistry. Viability of isolated pancreatic acinar cells in response to LO, KYNA, SZR-72 and/or NMDA administration was assessed by propidium-iodide assay. The effects of LO and/or SZR-72 on neutrophil granulocyte function was also studied. Almost all investigated laboratory and histological parameters of AP were significantly reduced by administration of 300 mg/kg KYNA or SZR-72, whereas the 150 mg/kg or 75 mg/kg doses were less or not effective, respectively. The decreased pancreatic microcirculation was also improved in the AP groups treated with 300 mg/kg KYNA or SZR-72. Interestingly, pancreatic heat shock protein-72 expression was significantly increased by administration of SZR-72, KYNA and/or LO. mRNA and protein expression of NMDAR1 was detected in pancreatic tissue. LO treatment caused acinar cell toxicity which was reversed by 250 µM KYNA or SZR-72. Treatment of acini with NMDA (25, 250, 2000 µM) did not influence the effects of KYNA or SZR-72. Moreover, SZR-72 reduced LO-induced H2O2 production of neutrophil granulocytes. KYNA and SZR-72 have dose-dependent protective effects on LO-induced AP or acinar toxicity which seem to be independent of pancreatic NMDA receptors. Furthermore, SZR-72 treatment suppressed AP-induced activation of neutrophil granulocytes. This study suggests that administration of KYNA and its derivative could be beneficial in AP.

Acute pancreatitis (AP) is a potentially life-threatening gastrointestinal disease with a complex pathology including oxidative stress. Oxidative stress triggers oxidative DNA lesions such as formation of 7,8-dihydro-8-oxo-2′-oxoguanine (8-oxoG) and also causes DNA strand breaks. DNA breaks can activate the nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) which contributes to AP pathology. 8-oxoG is recognized by 8-oxoG glycosylase 1 (OGG1) resulting in the removal of 8-oxoG from DNA as an initial step of base excision repair. Since OGG1 also possesses a DNA nicking activity, OGG1 activation may also trigger PARP1 activation. In the present study we investigated the role played by OGG1 in AP. We found that the OGG1 inhibitor compound TH5487 reduced edema formation, inflammatory cell migration and necrosis in a cerulein-induced AP model in mice. Moreover, TH5487 caused 8-oxoG accumulation and reduced tissue poly(ADP-ribose) levels. Consistent with the indirect PARP inhibitory effect, TH5487 shifted necrotic cell death (LDH release and Sytox green uptake) towards apoptosis (caspase activity) in isolated pancreatic acinar cells. In the in vivo AP model, TH5487 treatment suppressed the expression of various cytokine and chemokine mRNAs such as those of TNF, IL-1β, IL1ra, IL6, IL16, IL23, CSF, CCL2, CCL4, CCL12, IL10 and TREM as measured with a cytokine array and verified by RT-qPCR. As a potential mechanism underlying the transcriptional inhibitory effect of the OGG1 inhibitor we showed that while 8-oxoG accumulation in the DNA facilitates NF-κB binding to its consensus sequence, when OGG1 is inhibited, target site occupancy of NF-κB is impaired. In summary, OGG1 inhibition provides protection from tissue injury in AP and these effects are likely due to interference with the PARP1 and NF-κB activation pathways.

Acute pancreatitis (AP) poses a worldwide challenge due to the growing incidence and its potentially life-threatening course and complications. Specific targeted therapies are not available, prompting the identification of new pathways and novel therapeutic approaches. Flavonoids comprise several groups of biologically active compounds with wide-ranging effects. The flavone compound, tricetin (TCT), has not yet been investigated in detail but sporadic reports indicate diverse biological activities. In the current study, we evaluated the potential protective effects of TCT in AP. TCT (30 μM) protected isolated primary murine acinar cells from the cytotoxic effects of cerulein, a cholecystokinin analog peptide. The protective effects of TCT were observed in a general viability assay (calcein ester hydrolysis), in an apoptosis assay (caspase activity), and in necrosis assays (propidium iodide uptake and lactate dehydrogenase release). The effects of TCT were not related to its potential antioxidant effects, as TCT did not protect against H2O2-induced acinar cell death despite possessing radical scavenging activity. Cerulein-induced expression of IL1β, IL6, and matrix metalloproteinase 2 and activation of nuclear factor-κB (NFκB) were reduced by 30 μM TCT. In vivo experiments confirmed the protective effect of TCT in a mouse model of cerulein-induced AP. TCT suppressed edema formation and apoptosis in the pancreas and reduced lipase and amylase levels in the serum. Moreover, TCT inhibited interleukin-1β (IL1β), interleukin-6 (IL6), and tumor necrosis factor-α (TNFα) expression in the pancreas and reduced the activation of the oxidative DNA damage sensor enzyme poly(ADP-ribose) polymerase-1 (PARP-1). Our data indicate that TCT can be a potential treatment option for AP.

Nitric oxide (NO) bioavailability is fundamental in the regulation of redox balance and functionality of the endothelium, especially in the case of the umbilical cord (UC), which has no innervation. The analysis of UC vessel-related complications could serve as a useful tool in the understanding of the pathophysiological mechanisms leading to neonatal cardiovascular disorders. Therefore, the aim of this study was to characterize the mechanisms that rule the severity of prenatal endothelial dysfunction, induced by the long-term effect of maternal smoking. Our analysis describes the initiation and the consequences of endothelial nitric oxide synthase (NOS3) deactivation, along with the up-regulation of possible compensatory pathways, using structural, molecular and biochemical approaches. This study was carried out on both the UC arteries and veins originated from neonates born to non-smoking and heavy-smoking mothers. The alterations stimulated by maternal smoking are vessel-specific and proportional to the level of exposure to harmful materials passed through the placenta. Typically, in the primarily exposed veins, an increased formation of reactive oxygen species and an up-regulation of the highly-efficient NOS2-NO producing pathway were detected. Despite all the extensive structural and functional damages, the ex vivo heat and cadmium ion-treated UC vein pieces still support the potential for stress response.

Gut microbial composition alters in some special situations, such as in ulcerative colits (UC) after total proctocolectomy and ileal pouch-anal anastomosis (IPAA) surgery. The aim of our study was to determine the composition of the intestinal microbiome in UC patients after IPAA surgery, compared with UC patients, familial adenomatous polyposis (FAP) patients after IPAA surgery and healthy controls. Clinical data of patients, blood and faecal samples were collected. Faecal microbiota structure was determined by sequencing the V4 hypervariable region of the 16S rRNA gene. Overall, 56 patients were enrolled. Compared to the Healthy group, both the Pouch active and UC active groups had higher Enterobacteriaceae, Enterococcaceae and Pasteurellaceae abundance. The Pouch and UC groups showed distinct separation based on their alpha and beta bacterial diversities. The UC group had higher Prevotellaceae, Rikenellaceae, Ruminococcaceae abundance compared to the Pouch active group. Pouch and FAP participants showed similar bacterial community composition. There was no significant difference in the bacterial abundance between the active and inactive subgroups of the Pouch or UC groups. Gut microbiome and anatomical status together construct a functional unit that has influence on diversity, in addition to intestinal inflammation that is a part of the pathomechanism in UC.

Cystic fibrosis (CF) is the most common genetic disorder that causes a significant damage in secretory epithelial cells due to the defective ion flux across the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Pancreas is one of the organs most frequently damaged by the disease leading to pancreatic insufficiency, abdominal pain and an increased risk of acute pancreatitis in CF patients causing a significant decrease in the quality of life. CFTR plays a central role in the pancreatic ductal secretory functions by carrying Cl- and HCO3- ions across the apical membrane. Therefore pathophysiological studies in CF mostly focused on the effects of impaired ion secretion by pancreatic ductal epithelial cells leading to exocrine pancreatic damage. However, several studies indicated that CFTR has a central role in the regulation of intracellular signaling processes and is now more widely considered as a signaling hub in epithelial cells. In contrast, elevated intracellular Ca2+ level was observed in the lack of functional CFTR in different cell types including airway epithelial cells. In addition, impaired CFTR expression has been correlated with damaged mitochondrial function in epithelial cells. These alterations of intracellular signaling in CF are not well characterized in the exocrine pancreas yet. Therefore in this review we would like to summarize the complex role of CFTR in the exocrine pancreas with a special focus on the intracellular signaling and mitochondrial function.

Acute pancreatitis (AP) is often accompanied by alterations in the acid-base balance, but how blood pH influences the outcome of AP is largely unknown. We studied the association between blood pH and the outcome of AP with meta-analysis of clinical trials, and aimed to discover the causative relationship between blood pH and AP in animal models. PubMed, EMBASE, and Cochrane Controlled Trials Registry databases were searched from inception to January 2017. Human studies reporting systemic pH status and outcomes (mortality rate, severity scores, and length of hospital stay) of patient groups with AP were included in the analyses. We developed a new mouse model of chronic metabolic acidosis (MA) and induced mild or severe AP in the mice. Besides laboratory blood testing, the extent of pancreatic edema, necrosis, and leukocyte infiltration were assessed in tissue sections of the mice. Thirteen studies reported sufficient data in patient groups with AP (n = 2,311). Meta-analysis revealed markedly higher mortality, elevated severity scores, and longer hospital stay in AP patients with lower blood pH or base excess (P < 0.001 for all studied outcomes). Meta-regression analysis showed significant negative correlation between blood pH and mortality in severe AP. In our mouse model, pre-existing MA deteriorated the pancreatic damage in mild and severe AP and, vice versa, severe AP further decreased the blood pH of mice with MA. In conclusion, MA worsens the outcome of AP, while severe AP augments the decrease of blood pH. The discovery of this vicious metabolic cycle opens up new therapeutic possibilities in AP.

Patients with recurrent acute pancreatitis (RAP) are at significant risk of developing early chronic pancreatitis (CP), which progresses into irreversible, end-stage CP with severe symptoms. There is no specific therapy in RAP or in early CP that may hinder disease progression. The pathogenesis of CP is complex and involves interactions among multiple cell types, including pancreatic acinar, ductal, and stellate cells (PSC). Therefore, it is pivotal to identify common pathogenic pathways in these cells that could be targeted pharmacologically. The Orai1-mediated store-operated Ca2+ entry (SOCE) is a ubiquitous signaling mechanism that may become overactivated in pathological states resulting in intracellular Ca2+ overload. In this study, we used ex vivo and in vivo preclinical disease models to demonstrate that Orai1 inhibition prevents progression of RAP and early CP. The selective Orai1 inhibitor CM5480 restored the expression of SOCE-associated regulatory factor in acinar cells, prevented uncontrolled Ca2+ elevation, protected acinar and ductal functions, mitigated immune cell infiltration, and diminished PSC activation, proliferation, and migration. We suggest that the overactivation of Orai1 is a crucial pathogenetic event in the progression of early CP and that inhibition of Orai1 could prevent the development of end-stage CP.

Background: In patients with inflammatory bowel disease (IBD), Crohn’s disease (CD), and ulcerative colitis (UC), numerous cases of exacerbations could be observed after colonoscopy, raising the possible pathogenetic effect of colonic microbiota alterations in IBD flare. Objectives: We aimed to investigate the changes in the fecal microbiota composition in IBD patients influenced by the bowel preparation with sodium picosulfate. Design: We enrolled patients with IBD undergoing bowel preparation for colonoscopy in the prospective cohort study. The control group (Con) comprised non-IBD patients who underwent colonoscopy. Clinical data, blood, and stool samples were collected before colonoscopy (timepoint A), 3 days later (timepoint B), and 4 weeks later (timepoint C). Methods: Disease activity and gut microbiota changes were assessed at each timepoint. Fecal microbiota structure – at family level – was determined by sequencing the V4 region of the 16S rRNA gene. Statistical analysis included differential abundance analysis and Mann–Whitney tests. Results: Forty-one patients (9 CD, 13 UC, and 19 Con) were included. After bowel preparation, alpha diversity was lower in the CD group than in the UC ( p = 0.01) and Con ( p = 0.02) groups at timepoint B. Alpha diversity was significantly higher in the UC group than in the CD and Con ( p = 0.03) groups at timepoint C. Beta diversity difference differed between the IBD and Con ( p = 0.001) groups. Based on the differential abundance analysis, the Clostridiales family was increased, whereas the Bifidobacteriaceae family was decreased in CD patients compared to the Con at timepoint B. Conclusions: Bowel preparation may change the fecal microbial composition in IBD patients, which may have a potential role in disease exacerbation after bowel cleansing.

Abstract A broad-spectrum reactive oxygen species (ROS)-scavenging hybrid material (CASCADE) was developed by sequential adsorption of heparin (HEP) and poly(L-lysine) (PLL) polyelectrolytes together with superoxide dismutase (SOD) and horseradish peroxidase (HRP) antioxidant enzymes on layered double hydroxide (LDH) nanoclay support. The synthetic conditions were optimized so that CASCADE possessed remarkable structural (no enzyme leakage) and colloidal (excellent resistance against salt-induced aggregation) stability. The obtained composite was active in decomposition of both superoxide radical anions and hydrogen peroxide in biochemical assays revealing that the strong electrostatic interaction with the functionalized support led to high enzyme loadings, nevertheless, it did not interfere with the native enzyme conformation. In vitro tests demonstrated that ROS generated in human cervical adenocarcinoma cells were successfully consumed by the hybrid material. The cellular uptake was not accompanied with any toxicity effects, which makes the developed CASCADE a promising candidate for treatment of oxidative stress-related diseases.

Objective: Defective mucus production in the pancreas may be an important factor in the initiation and progression of chronic pancreatitis (CP), therefore we aimed to (i) investigate the qualitative and quantitative changes of mucus both in human CP and in an experimental pancreatitis model and (ii) to correlate the mucus phenotype with epithelial ion transport function.Design: Utilizing human tissue samples and a murine model of cerulein induced CP we measured pancreatic ductal mucus content by morphometric analysis and the relative expression of different mucins in health and disease. Pancreatic fluid secretion in CP model was measured in vivo by magnetic resonance cholangiopancreatography (MRCP) and in vitro on cultured pancreatic ducts. Time-changes of ductal secretory function were correlated to those of the mucin production.Results: We demonstrate increased mucus content in the small pancreatic ducts in CP. Secretory mucins MUC6 and MUC5B were upregulated in human, Muc6 in mouse CP. In vivo and in vitro fluid secretion was decreased in cerulein-induced CP. Analysis of time-course changes showed that impaired ductal ion transport is paralleled by increased Muc6 expression.Conclusion: Mucus accumulation in the small ducts is a combined effect of mucus hypersecretion and epithelial fluid secretion defect, which may lead to ductal obstruction. These results suggest that imbalance of mucus homeostasis may have an important role in the early-phase development of CP, which may have novel diagnostic and therapeutic implications.

Biomedical investment trends in 2015 show a huge decrease of investment in gastroenterology. Since academic research usually provides the basis for industrial research and development (R&D), our aim was to understand research trends in the field of gastroenterology over the last 50 years and identify the most endangered areas.We searched for PubMed hits for gastrointestinal (GI) diseases for the 1965-2015 period. Overall, 1,554,325 articles were analyzed. Since pancreatology was identified as the most endangered field of research within gastroenterology, we carried out a detailed evaluation of research activity in pancreatology.In 1965, among the major benign GI disorders, 51.9% of the research was performed on hepatitis, 25.7% on pancreatitis, 21.7% on upper GI diseases and only 0.7% on the lower GI disorders. Half a century later, in 2015, research on hepatitis and upper GI diseases had not changed significantly; however, studies on pancreatitis had dropped to 10.7%, while work on the lower GI disorders had risen to 23.4%. With regard to the malignant disorders (including liver, gastric, colon, pancreatic and oesophageal cancer), no such large-scale changes were observed in the last 50 years. Detailed analyses revealed that besides the drop in research activity in pancreatitis, there are serious problems with the quality of the studies as well. Only 6.8% of clinical trials on pancreatitis were registered and only 5.5% of these registered trials were multicentre and multinational (more than five centres and nations), i.e., the kind that provides the highest level of impact and evidence level.There has been a clear drop in research activity in pancreatitis. New international networks and far more academic R&D activities should be established in order to find the first therapy specifically for acute pancreatitis.